C31 | Antimicrobial resistance in the environment and wastewater of a pig slaughterhouse – monitoring of ESKAPE bacteria and ESBL/carbapenemase-producing Escherichia coli

C. Manfreda¹, F. Guarneri², G. Ferri³, C. Di Vittori³, A. Olivastri⁴, M.A. Piccioni⁴, A. Ianieri¹, G.L. Alborali², A. Vergara³. | ¹Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Parma; ²Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna “B. Umbertini”, Brescia; ³Scuola di Specializzazione in Ispezione degli Alimenti di Origine Animale “G. Tiecco”, Dipartimento di Medicina Veterinaria, Università degli Studi di Teramo, Teramo; ⁴Servizio Veterinario I.A.O.A., AST Marche, Area Vasta 5, Ascoli Piceno.

Abstract

Purpose. The purpose of this study was to investigate the phenomenon of antimicrobial resistance in ESKAPE bacterial strains and ESBL/carbapenemase-producing Escherichia coli isolated and identified from environmental samples and wastewater from a pig slaughterhouse. Methods. Between January and May 2025, six inspections were carried out at a slaughterhouse in central Italy, and 48 environmental and wastewater samples were collected. Specifically, 24 samples were taken from the floors of the scalding tank area and the cutting area during slaughtering operations and after cleaning and disinfection (ISO 18593:2018). The remaining 24 samples were wastewater (1.0 L aliquots) sampled before and after the purification process. Microbiological analyses included: methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Klebsiella pneumoniae and E. coli indicators and ESBL/carbapenemase producers, and total bacterial count (TBC). Strain identification was performed using MALDI-TOF, except for E. coli, which was typed using Clermont. All strains were tested for sensitivity to 18 antimicrobial molecules, obtaining the minimum inhibitory concentration (MIC test - EUCAST breakpoints). Finally, multiplex end-point PCR assays were used to amplify resistance genes (ARGs) associated with ESBL (bla_CTX-M, bla_SHV, bla_TEM and bla_CMY) and carbapenemase (bla_VIM, bla_IMP, bla_KPC, bla_NDM, bla_OXA-48 and bla_GES-5). 3. Results. Microbiological screening allowed the isolation and identification of a total of 53 strains, originating from 64.58% of the samples analysed. More specifically, 39/53 (73.58%) Escherichia coli were isolated, 71.79% of which came from water (57.14% before and 42.86% after purification) and 28.20% from the surfaces tested. Finally, 3 E. coli (3/39 – 7.69%) were found to be ESBL producers (isolated from the floor during operation). Next, 7 (7/53 - 13.20%) strains of Acinetobacter spp. were identified, 57.14% from pre-purification water, and finally, 6 (6/53 - 11.32%) strains of S. aureus, of which 2 were methicillin-resistant (MRSA) from the floor during the operational phases. From the phenotypic resistance profiles observed, it was found that 61.54% (24/39) of E. coli was resistant to at least one of the molecules tested; of these, 50.0% (12/24) was multi-resistant. The prevalences found were as follows: 75.00% ampicillin, 66.67% tetracycline, 54.17% trimethoprim and 37.50% chloramphenicol. The three ESBL strains were resistant to cefepime and cefotaxime, and the ARGs bla_CTX-M and bla_TEM were amplified from them. Among the Acinetobacter spp., 71.43% were resistant to the combination of ceftazidime/clavulanic acid, and finally, the MRSA strains were resistant to at least seven antimicrobial molecules. The CBT showed an average value of 5.77 log CFU/cm². Conclusions. The results obtained demonstrated the presence of ESKAPE bacteria and ESBL E. coli in the slaughterhouse ecosystem and wastewater. The environment is therefore a key variable in the study and understanding of antimicrobial resistance and the management of resistant microorganisms.